Platelet-derived Growth Factor-induced H sub(2)O sub(2) Production Requires the Activation of Phosphatidylinositol 3-Kinase

Autophosphorylation of the platelet-derived growth factor (PDGF) receptor triggers intracellular signaling cascades as a result of recruitment of Src homology 2 domain-containing enzymes, including phosphatidylinositol 3-kinase (PI3K), the GTPase-activating protein of Ras (GAP), the protein-tyrosine phosphatase SHP-2, and phospholipase C- gamma 1 (PLC- gamma 1), to specific phosphotyrosine residues. The roles of these various effectors in PDGF-induced generation of H sub(2)O sub(2) have now been investigated in HepG2 cells expressing various PDGF receptor mutants. These mutants included a kinase-deficient receptor and receptors in which various combinations of the tyrosine residues required for the binding of PI3K (Tyr super(740) and Tyr super(751)), GAP (Tyr super(771)), SHP-2 (Tyr super(1009)), or PLC- gamma 1 (Tyr super(1021)) were mutated to Phe. PDGF failed to increase H sub(2)O sub(2) production in cells expressing either the kinase-deficient mutant or a receptor in which the two Tyr residues required for the binding of PI3K were replaced by Phe. In contrast, PDGF-induced H sub(2)O sub(2) production in cells expressing a receptor in which the binding sites for GAP, SHP-2, and PLC- gamma 1 were all mutated was slightly greater than that in cells expressing the wild-type receptor. Only the PI3K binding site was alone sufficient for PDGF-induced H sub(2)O sub(2) production. The effect of PDGF on H sub(2)O sub(2) generation was blocked by the PI3K inhibitors LY294002 and wortmannin or by overexpression of a dominant negative mutant of Rac1. These results suggest that a product of PI3K is required for PDGF- induced production of H sub(2)O sub(2) in nonphagocytic cells, and that Rac1 mediates signaling between the PI3K product and the putative NADPH oxidase.